Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Schematic overview of the IgE elimination experiment. Humanized FcγRIIb mice ( b – e ; n = 5 mice per group) or FcγRIIb -/- mice ( f , g ; n = 4 mice per group) were injected with 5 mg/kg FcRTACs,followed by injection of 2.5 mg/kg IgE 30 min later. Blood samples were collected at the indicated time points (red arrows) to measure the serum levels of total IgE ( b , d , f , g ) and free IgE ( c , e ). Data are presented as means ± SEM. Statistical significance was determined using two-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ). The curves for “Only IgE”, “Omalizumab”, and “NK-2-12” are shared between panels b and d , as well as c and e . Panel ( a ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/53ug670 .

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Injection

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Alignment of SK3 and SK4 Sequences. HCDRs and LCDRs are indicated with red. b SPR-based competition assay for SK3 and SK4. NK-2-12-SK3 was immobilized on a protein A biosensor chip, followed by injection of hFcγRIIb over the flow cell. Subsequently, SK4 Fab and hFcγRIIb were injected. c Structure of the SK4 Fab in complex with FcγRIIb (PDB ID: 9M5B ). The heavy and light chains of the SK4 Fab are shown in orange and light blue, respectively. The ribbon diagram of FcγRIIb is colored green, with its surface illustrated in white and the contact area with SK4 Fab highlighted in brown. d Comparison of the calculated SK3 scFv-FcγRIIb complex structure versus the SK4 Fab-FcγRIIb complex structure. e SPR-based affinity measurements of SK3 and SK3hi binding to hFcγRIIb at pH 7.4 and pH 6.0. SK3 or SK3hi was immobilized on a protein A biosensor chip, followed by injection of hFcγRIIb over the flow cell. Solid curves represent raw data, while dashed curves indicate fitted curves. f Live-cell imaging of NK-2-12-SK3hi-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells in the presence or absence of Cy5-labeled NK-2-12-SK3hi (white) for 2 h. Cells were stained with LysoTracker (green) and Hoechst (blue) for confocal imaging. Images were acquired using a super-resolution confocal microscope. Scale bar: 5 μm. Experiments were performed in triplicate and repeated three times with similar results. g , h The rechallenge experiment. FcγRIIb humanized mice ( n = 4 mice per group) were i.p. injected with 2.5 mg/kg IgE following by injection of 2.5 mg/kg NK-2-12-SK3 or NK-2-12-SK3hi. One day later, mice were rechallenged with 2.5 mg/kg IgE without FcRTACs. Blood samples were collected to measure serum IgE levels. Data are presented as means ± SEM. Statistical comparisons were performed using Mixed-effects analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ).

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Competitive Binding Assay, Injection, Comparison, Binding Assay, Live Cell Imaging, Labeling, Incubation, Staining, Imaging, Microscopy

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Schematic diagram of the structure of NK-2-12-v12. b Internalization assay of NK-2-12-V12. Antibodies were pre-mixed with a pH-sensitive dye-labeled anti-human Fc and incubated with Huvec-2b cells. Fluorescence changes were monitored hourly over 24 h. c , Internalization assay of NK-2-12-V12 in presence of endocytosis inhibitors. d , e Live-cell imaging of bispecific antibodies-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells in the presence of Cy5-labeled designated antibodies (white) for 1 h. Cells were stained with LysoTracker (green) and Hoechst (blue) for confocal imaging. Images were acquired using a 40× objective ( d , scale bar: 40 μm) or a 60× objective confocal microscope ( e left panel, scale bar: 20 μm) and a super-resolution microscope ( e middle and right panel; scale bar: 10 μm). Experiments were performed in triplicate and repeated three times with similar results. f FcγRIIb humanized mice were injected with 5 mg/kg NK-2-12-SK3 or NK-2-12-V12, followed by administration of 2.5 mg/kg IgE after 30 min. Blood samples were collected to measure the serum IgE levels ( n = 4 mice per group). Data are presented as means ± SEM. Statistical significance was determined using two-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ). The curves for “Only IgE” is shared between Supplementary Fig. . g FcγRIIb humanized mice were injected with 5 mg/kg of NK-2-12-SK3 and NK-2-12-V12. Blood was collected from the tail vein of mice at indicated time points and serum antibody levels were quantitatively detected by ELISA ( n = 5 mice per group). Panel ( a ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/g5wqarw .

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Labeling, Incubation, Fluorescence, Live Cell Imaging, Staining, Imaging, Microscopy, Super-Resolution Microscopy, Injection, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Virus envelope glycoprotein targeting bispecific T cell engager protects mice from lethal severe fever with thrombocytopenia virus infection

doi: 10.1016/j.xcrm.2025.102458

Figure Lengend Snippet: Development of BiTEs and evaluation of the efficacy in treating SFTSV infection (A) The design of anti-Gn/anti-CD3 BiTEs. (B) Reduced SDS-PAGE analysis of purified anti-Gn/anti-CD3 BiTEs. (C) The ability of BiTEs to bind to CD3 and Gn simultaneously is determined by a surface plasmon resonance experiment. CD3E&CD3D heterodimer protein is captured on a Series S CM5 chip by amine coupling followed by sequential injection of bispecific antibodies and the Gn-Fc fusion protein. (D) Relative inhibition rates determined by comparing SFTSV-NP-positive Huh-7 cells using flow cytometry ( n = 3 biologically independent replicates). Huh-7 cells were infected with SFTSV (MOI = 0.2) and then co-cultured with T cells in the presence of BiTEs for 48 h. (E and F) Relative inhibition rates determined by comparing SFTSV NP expression in Huh-7 cells using flow cytometry ( n = 3 biologically independent replicates). Huh-7 cells were infected with SFTSV (MOI = 0.2) and then co-cultured with T cells in the presence of 3A5 BiTE along with either an anti-IFNG antibody, a GZMB inhibitor, or a perforin inhibitor for 48 h. T cells were purified from two independent donors (donors 1 and 2). (G) A graphical representation of the in vivo study design for evaluating the protective efficacy of BiTE against SFTSV infection. The humanized CD3e C57BL/6 mice were pretreated with anti-IFNAR1 antibody and then intraperitoneally inoculated with SFTSV (4 × 10 4 PFUs per mouse) one day later. Two hours after infection, mice were intraperitoneally injected with 3A5 BiTE, MAb4-5 BiTE, control BiTE (10 mg/kg for each mouse) or an equal volume of PBS. (H) Survival curves of mice treated with BiTE or PBS ( n = 6 per group) after SFTSV challenge. Death is defined as a humane endpoint when animals lost ≥20% body weight but not natural death. (I–L) Viral loads in serum (I) and viral titers in spleen (J), liver (K), and lung (L) samples from mice ( n = 6 per group) measured at 6 days post-infection (dpi) by RT-qPCR and immunological focus assay, respectively. (M) Representative images of spleens collected at 6 dpi from uninfected mice and SFTSV-challenged mice with BiTE or PBS treatment. (N) Representative images of three replicates of spleen sections collected at 6 dpi from uninfected mice and SFTSV-challenged mice with BiTE or PBS treatment stained with hematoxylin and eosin. Black arrows indicate megakaryocytes. Scale bars, 500 μm. See also . Data were presented as mean ± SD (D–F and I–L). One-way ANOVA followed by Tukey’s multiple comparisons test was performed for comparison of continuous variables among multiple groups (D–F and I–L). The Kaplan-Meier method was used to analyze time-to-event data (H). ∗ p < 0.05; ∗∗ p < 0.01; ns: no significance.

Article Snippet: Six-week-old C57BL/6J were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and humanized CD3e C57BL/6 mice (Cat. NO. NM-HU-00114) were purchased from Shanghai Model Organisms Center, Inc. Mice were housed under specific-pathogen-free conditions with daily cycles of 12 h light/12 h darkness, temperature of 22°C, and humidity of 50%.

Techniques: Infection, SDS Page, Purification, SPR Assay, Injection, Inhibition, Flow Cytometry, Cell Culture, Expressing, In Vivo, Control, Quantitative RT-PCR, Staining, Comparison

Journal: Cell Reports Medicine

Article Title: Virus envelope glycoprotein targeting bispecific T cell engager protects mice from lethal severe fever with thrombocytopenia virus infection

doi: 10.1016/j.xcrm.2025.102458

Figure Lengend Snippet: BiTEs provided treatment effects against lethal SFTSV infection through improving T cell cytotoxicity in vivo (A) Percentage of CD45 + T cells, CD4 + T cells, CD8 + T cells, CD19 + B cells, CD11b + Gr-1 + neutrophils, and Foxp3 + Tregs in spleen samples collected at 6 dpi from mice ( n = 5 per group) measured by flow cytometry. (B) A schematic diagram showing depletion of T cells in mice to determine the role of T cells in 3A5 BiTE treatment. Six- to eight-week-old male humanized CD3e C57BL/6 mice were pretreated with anti-IFNAR1 antibody (300 μg per mouse) and then intraperitoneally inoculated with SFTSV (2 × 10 4 PFUs per mouse) one day later. One day after viral infection, mice were intraperitoneally injected with 3A5 BiTE (10 mg/kg for each mouse) or an equal volume of PBS. Anti-mouse CD4 and CD8a antibodies were used for depletion of T cells. (C) Survival curves of SFTSV-challenged mice treated with PBS ( n = 12) or 3A5 BiTE ( n = 12) and mice treated with 3A5 BiTE and depleted with CD4 + or CD8 + T cells ( n = 7). Death is defined as a humane endpoint when animals lost ≥20% body weight but not natural death. (D) Viral titers in spleen samples from mice measured by immunological focus assay, at 6 days post-infection (dpi). (E) Representative images of spleen sections collected at 6 dpi stained with hematoxylin and eosin (H&E) or with an antibody against SFTSV NP. Scale bars, 100 μm. See also . Data were presented as mean ± SD (A and D). One-way ANOVA followed by Tukey’s multiple comparisons test was performed for comparison of continuous variables among multiple groups (A and D). The Kaplan-Meier method was used to analyze time-to-event data (C). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns: no significance.

Article Snippet: Six-week-old C57BL/6J were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and humanized CD3e C57BL/6 mice (Cat. NO. NM-HU-00114) were purchased from Shanghai Model Organisms Center, Inc. Mice were housed under specific-pathogen-free conditions with daily cycles of 12 h light/12 h darkness, temperature of 22°C, and humidity of 50%.

Techniques: Infection, In Vivo, Flow Cytometry, Injection, Staining, Comparison